The present invention is directed to peptide inhibitors of angiogenesis. More specifically, this invention relates to a peptide that interacts with thrombospondin 2 and inhibits matrix metalloproteinase 9 (MMP-9) activity.
Matrix metalloproteinases, or MMPs, are a family of zinc proteinases that digest the extracellular matrix and are implicated in a variety of pathological conditions, including early stages of angiogenesis, cardiovascular diseases, and cancer metastasis. Compounds that inhibit MMP activity are thought to be useful for the treatment or prophylaxis of conditions that involve tissue breakdown. These include rheumatoid arthritis, osteoarthritis, gastric ulceration, cancer metastasis as well as management of angiogenesis-dependent diseases. There is growing body of evidence suggesting that anti-angiogenic inhibitors will improve future therapies of diseases such as cancer, rheumatoid arthritis and ocular neovascularization. Important therapeutic strategies include suppression of activity of the major angiogenic regulators such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF); inhibition of activity of alphav-integrins and matrix metalloproteinases (MMPs); and the exploitation of endogenous anti-angiogenic molecules like angiostatin, endostatin or thrombospond.
Matrix metalloproteinases or MMPs, including MMP-2 and MMP-9, are expressed in a variety of tissues. Matrix metalloproteinase 9 degrades Type IV collagen, a major component of extracellular matrix and is believed to be crucial for cancer invasion, metastasis and angiogenesis.
Angiogenesis is the process of new blood vessel development and formation and plays an important role in numerous physiological events, both normal and pathological. Angiogenesis occurs in response to specific signals and involves a complex process characterized by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signal(s), migration of the endothelial cells toward the source of the signal(s), and subsequent proliferation and formation of the capillary tube. Blood flow through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization, including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., 1991, Biotech. 9:630-634; Folkman et al., 1995, N. Engl. J. Med., 333:1757-1763; Auerbach et al., 1985, J. Microvasc. Res. 29:401-411; Folkman, 1985, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; and Folkman et al., 1983, Science 221:719-725.
In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggests that the growth of solid tumors and lethality are dependent on angiogenesis. Folkman and Klagsbrun, 1987, Science 235:442-447. The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., 1989, Cell 56:345-355. In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as those characterizing solid tumor growth, these regulatory controls fail.
Thrombospondins are naturally occurring inhibitors of angiogenesis. However, the molecular mechanisms by which they function are not well understood. Thrombospondins (TSP) are a family of extracellular matrix glycoproteins that include at least five related members (TSP 1-5). Thrombospondins are thought to play a role in a number of biological processes including coagulation, cell adhesion, cell growth, modulation of cellxe2x80x94cell and cell-matrix interactions, control of tumor growth and metastases, and angiogenesis. Thrombospondin-1 (TSP-1) is the most extensively studied thrombospondin to date. TSP-1 is capable of inhibiting neovascularization induced by angiogenic factors. In addition, TSP-1 has been shown to inhibit angiogenesis both in vitro and in vivo. The role of Thrombospondin-2 (TSP-2) in angiogenesis, however, is not very well understood.
Thrombospondin-1 and Thrombospondin-2 share many structural properties. Both contain N- and C-terminal globular domains flanking a procollagen homology region, three properdin-like type 1 repeats (TSR), three EGF-like type 2 repeats and seven Ca2+ binding type 3 repeats. Structure-function studies have localized the anti-angiogenic region of TSP-1 to the procollagen-like region and the TSR. Thrombospondin-1 and Thrombospondin-2 share a high degree of homology in the TSR region. The spatial and temporal expression of TSP-1 and TSP-2 is very different both during embryonic development and in adult tissues. However similar to TSP-1, TSP-2 is highly expressed in developing blood vessels. Previously, it was reported that the TSR domains of both TSP-1 and TSP-2 interact with the N-terminal region of MMP-2, resulting in the inhibition of MMP catalytic activity. Although, both TSP-1 and TSP-2 are thought to elicit anti-angiogenic properties, TSP-1 and TSP-2 knockout mice exhibit different phenotypes suggesting different mechanisms of action. TSP-1 knockout mice show abnormalities primarily in the lungs with an increase in neutrophils and macrophages, suggestive of diffuse alveolar hemorrhage. The lack of TSP-2 expression in the presence of normal levels of TSP-1 expression is associated with disordered collagen fibrillogenesis and an increase in blood vessel count in the skin and other tissues. Furthermore, the tsp-2xe2x88x92/xe2x88x92 mice demonstrated accelerated angiogenesis. These and other changes are consistent with increased MMP activity. Although, the tsp-2xe2x88x92/xe2x88x92 mice show phenotypic changes that are consistent with increased MMP activity, the mechanism(s) by which TSP-2 inhibits MMP activity are not known. Understanding the mechanisms underlying the anti-angiogenic properties of TSP will aid in the exploitation of these molecules in the treatment of various angiogenesis related diseases and potentially diseases that show increased MMP activity.
This invention provides peptides that inhibit MMP-9 and are useful as inhibitors of angiogenesis and metastasis, among other activities regulated by MMP-9. More specifically, the present invention provides peptides that interact with the TSR domain of thrombospondins, especially TSP-2, and inhibit MMP-9 activity. As a consequence, peptides of the invention, and their methods of use, are therapeutics against diseases, such as cancer and arthritis, which are characterized by neovascularization and tissue break down.
The invention provides a link between thrombospondins and matrix metalloproteinases and is based upon the discovery of a novel class of peptide inhibitors of MMP-9. Inhibitory peptides of the invention bind to the TSR domain of thrombospondins and cause a reduction in MMP-9 activation and its attendant effects on angiogenesis. Exemplary of this class of peptides is the peptide shown in SEQ ID NO:1. According to the invention, this peptide binds to the TSR domain of TSP-2 and inhibits MMP-9 activity. The invention also provides analogs, homologs and fragments of the peptide shown in SEQ ID NO:1 (APWNCCPVSCCGNCCFFRAL QSCPQG) which maintain the ability to bind to TSP-2 and to inhibit MMP-9 activity. The invention further provides nucleic acids encoding the peptides described above. In particular, in a preferred embodiment, a nucleic acid of the invention is shown in SEQ ID NO:2 (5xe2x80x2 GCGCC ATGGAACTGCTGTCCCGTGTCCTGCTGTGGAAACTGCTGCTTCTTCAGAGCTCTGCAGTC CTGTCCTCAGGGA).
The invention also comprises antibodies to epitope(s) encoded by the peptide or fragments of the peptide in SEQ ID NO:1. These antibodies are either polyclonal or monoclonal. The antibodies are made according to techniques known in the art.
This invention further comprises vectors containing the nucleic acid in SEQ ID NO:2 operably linked to regulatory DNA 5xe2x80x2 or 3xe2x80x2 of the nucleic acid of SEQ ID NO:2, that results in the translation of the nucleic acid into the peptide of SEQ ID NO:1. Regulatory DNA preferably comprises a promoter which is either a prokaryotic or a eukaryotic promoter, in order to facilitate expression in prokaryotic and eukaryotic cells respectively. In addition, vectors may contain the nucleic acid in SEQ ID NO:2 linked in-frame to another piece of DNA which results in expression of a fusion protein containing the peptide in SEQ ID NO:1 or homologs, fragments and variants of the peptide of this invention.
A preferred embodiment of the invention is a prokaryotic cell carrying the nucleic acid in SEQ ID NO:2 expressing the peptide in SEQ ID NO:1 or a fragment or variant or homolog that retains the MMP-9 inhibitory activity; a vector expressing the peptide or a fusion protein containing the peptide in SEQ ID NO:1 or a fragment or variant or homolog of the peptide.
Another preferred embodiment of the invention is a eukaryotic cell carrying the nucleic acid in SEQ ID NO:2 expressing the peptide in SEQ ID NO:1 or a fragment or variant or homolog that retains the MMP-9 inhibitory activity; a vector expressing the peptide or a fusion protein containing the peptide in SEQ ID NO:1 or a fragment or variant or homolog of the peptide.
Pharmaceutical compositions of peptide or fragments, variants or homologs of the peptide may be used to inhibit the action of MMP-9, for example, in the treatment or prophylaxis of disorders characterized by degradation of the extracellular matrix, such as, for example, cancer, arthritis and, cardiovascular disorders. Compositions of the invention are provided to an animal by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally). Where the compositions of the invention are to be provided parenterally, such as by intravenous, subcutaneous, opthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intranasal or by aerosol administration, the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution.